EPHB4对骨肉瘤细胞恶性进展的调控作用及机制研究

doi:10.3870/j.issn.1672-8009.2026.01.007

摘要/Abstract

摘要： 目的探讨肝配蛋白B4(erythropoietin-producing hepatocyte receptor B4,EPHB4)对骨肉瘤恶性进展的调控与作用机制。方法蛋白质印迹法检测U2OS、SaOS2-LM7、SaOS2、143B中EPHB4的表达变化。将SaOS2-LM7分为对照组、si-NC组、si-EPHB4组、sEPHB4(可溶性EPHB4封闭抗体)组,利用CCK-8法和EdU染色检测细胞增殖,利用流式细胞术检测细胞凋亡,利用Transwell实验和细胞划痕实验检测细胞侵袭迁移能力。用红色荧光蛋白(red fluorescent protein,RFP)标记的SaOS2-LM7构建稳定沉默EPHB4的细胞(EPHB4-si-RNA-RFP)。在体内实验中,将24只荷瘤裸鼠分为2组,即control-RFP组和EPHB4-siRNA-RFP组。通过小动物活体成像仪检测1~4周后癌细胞的肺转移情况。免疫组织化学法分析4周后肿瘤组织中EPHB4、分化簇33(cluster of differentiation 33,CD33)、细胞周期蛋白D1(CYCLIN D1)、基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)、MMP-14、β-CATENIN的表达。结果与U2OS比较,EPHB4在SaOS2-LM7、SaOS2、143B中的表达增加(P_均<0.05)。SaOS2-LM7细胞实验中,与对照组比较,sEPHB4组和si-EPHB4组的EPHB4表达、细胞的增殖活力和EdU阳性率显著降低,凋亡率显著增加,迁移率和侵袭率显著降低(P_均<0.05)。此外,与对照组比较,sEPHB4组和si-EPHB4组的β-CATENIN、CYCLIN D1、MMP-9、MMP-14的相对表达水平也显著降低(P_均<0.05)。在体内实验中,EPHB4-siRNA-RFP组在3周和4周的肺部转移率显著低于control-RFP组(P_均<0.05)。EPHB4-siRNA-RFP组的肿瘤组织中EPHB4、CD33、CYCLIN D1、MMP-9、MMP-14、β-CATENIN的表达均下调(P_均<0.05)。结论沉默EPHB4能显著抑制骨肉瘤细胞中WNT/β-CATENIN信号通路的激活,抑制细胞的增殖、迁移和侵袭,促进细胞凋亡。

关键词: 肝配蛋白B4, 骨肉瘤, 迁移, 侵袭, 凋亡, WNT/β-CATENIN信号通路

Abstract: Objective To explore the effect of erythropoietin-producing hepatocyte receptor B4(EPHB4)on the malignant progression of osteosarcoma and its mechanism. Methods The expression level of EPHB4 in U2OS,SaOS2-LM7,SaOS2,and 143B was detected by Western blotting.SaOS2-LM7 cells was divided into 4 groups:control group,si-NC group,si-EPHB4 group,and sEPHB4(soluble EPHB4 blocking antibody)group.Cell proliferation was detected by CCK-8 assay and EdU staining,cell apoptosis was detected by flow cytometry,cell invasion and migration were detected by Transwell assay and Wound healing assay.A SaOS2-LM7 cell line labeled with red fluorescent protein(RFP)which was stably silenced of EPHB4 was constructed(EPHB4-si-RNA-RFP).Twenty-four tumor-bearing nude mice were divided into 2 groups:the control-RFP group and the EPHB4-si-RNA-RFP group.The lung metastasis was detected 1-4 weeks later by using small animal in vivo imaging system.Immunohistochemistry was used to analyze the expression of EPHB4,cluster of differentiation 33(CD33),CYCLIN D1,matrix metalloproteinase 9(MMP-9),MMP-14,and β-CATENIN in tumor tissues 4 weeks later. Results The expression level of EPHB4 in the SaOS2-LM7,SaOS2,and 143B was significantly higher than that in the U2OS(all P<0.05).Compared with those in the control group,the expression level of EPHB4,the proliferation activity,and the EdU positive rate of SaOS2-LM7 in the sEPHB4 group and the si-EPHB4 group were significantly decreased,the apoptosis rate was significantly increased,and the migration rate and invasion rate were significantly decreased(all P<0.05).In addition,the relative expression levels of β-CATENIN,CYCLIN D1,MMP-9,and MMP-14 in the sEPHB4 group and the si-EPHB4 group were also significantly decreased when compared with those in the control group(all P<0.05).The lung metastasis at 3 weeks and 4 weeks was less in the EPHB4-siRNA-RFP group than in the control-RFP group(both P<0.05).The protein expression of EPHB4,CD33,CYCLIN D1,MMP-9,MMP-14,and β-CATENIN in the tumor tissues of the EPHB4-siRNA-RFP group was down-regulated(all P<0.05). Conclusion Silencing EPHB4 significantly inhibits the activation of the WNT/β-CATENIN signaling pathway in osteosarcoma cells,suppresses cell proliferation,migration,and invasion,and promotes cell apoptosis.

Key words: erythropoietin-producing hepatocyte receptor B4, osteosarcoma, migration, invasion, apoptosis, WNT/β-CATENIN signaling pathway

中图分类号:

R738.1

张飞, 陆红祥, 买买提克里木·吐松江, 杨童磊, 刘秋宏, 许刚. EPHB4对骨肉瘤细胞恶性进展的调控作用及机制研究[J]. 医学分子生物学杂志, 2026, 23(1): 51-59.

ZHANG Fei, LU Hongxiang, Maimaitikelimu Tusongjiang, YANG Tonglei, LIU Qiuhong, XU Gang. Regulation of EPHB4 on Malignant Progression of Osteosarcoma Cells[J]. Journal of Medical Molecular Biology, 2026, 23(1): 51-59.

参考文献

[1] EATON B R,SCHWARZ R,VATNER R,et al.Osteosarcoma[J].Pediatric Blood Cancer,2021,68(Suppl 2):e28352.
[2] JIANG J,PAN H,LI M,et al.Predictive model for the 5-year survival status of osteosarcoma patients based on the SEER database and XGBoost algorithm[J].Sci Rep,2021,11(1):5542.
[3] LILIENTHAL I,HEROLD N.Targeting molecular mechanisms underlying treatment efficacy and resistance in osteosarcoma:A review of current and future strategies[J].Int J Mol Sci,2020,21(18):6885.
[4] LI S,ZHANG H,LIU J,et al.Targeted therapy for osteosarcoma:a review[J].J Cancer Res Clin Oncol,2023,149(9):6785-67797.
[5] SU Q,WANG J,WU Q,et al.Sanguinarine combats hypoxia-induced activation of EPHB4 and HIF-1α pathways in breast cancer[J].Phytomedicine,2021,84:153503.
[6] NANAMIYA R,SAITO-KOYAMA R,MIKI Y,et al.EPHB4 as a novel target for the EGFR-independent suppressive effects of osimertinib on cell cycle progression in non-small cell lung cancer[J].Int J Mol Sci,2021,22(16):8522.
[7] XU C,GU L,KUERBANJIANG M,et al.Adaptive activation of EFNB2/EPHB4 axis promotes post-metastatic growth of colorectal cancer liver metastases by LDLR-mediated cholesterol uptake[J].Oncogene,2023,42(2):99-112.
[8] BHATIA S,NGUYEN D,DARRAGH L B,et al.EPHB4 and ephrinB2 act in opposition in the head and neck tumor microenvironment[J].Nat Commun,2022,13(1):3535.
[9] ZHU M,GONG Z,WU Q,et al.Homoharringtonine suppresses tumor proliferation and migration by regulating EPHB4-mediated β-CATENIN loss in hepatocellular carcinoma[J].Cell Death Dis,2020,11(8):632.
[10] FULORIA S,YADAV G,MENON S V,et al.Targeting the WNT/β-CATENIN cascade in osteosarcoma:The potential of ncRNAs as biomarkers and therapeutics[J].Pathol Res Pract,2024,259:155346.
[11] ARORA S,SCOTT A M,JANES P W.Eph receptors in cancer[J].Biomedicines,2023,11(2):315.
[12] ULLAH A,RAZZAQ A,ZHOU C,et al.Biological significance of EPHB4 expression in cancer[J].Curr Protein Pept Sci,2024,25(3):244-255.
[13] SCALIA P,MAURANCY J,GIORDANO A,et al.Transcriptional and post-translational control mechanisms for EPHB4 expression in physiology and cancer disease[J].Crit Rev Eukaryot Gene Expr,2021,31(2):83-88.
[14] YAN D,CUI D,ZHU Y,et al.M6PR- and EPHB4-Rich exosomes secreted by serglycin-overexpressing esophageal cancer cells promote cancer progression[J].Int J Biol Sci,2023,19(2):625-640.
[15] ZHANG Y,LIU Z,YANG X,et al.H3K27 acetylation activated-COL6A1 promotes osteosarcoma lung metastasis by repressing STAT1 and activating pulmonary cancer-associated fibroblasts[J].Theranostics,2021,11(3):1473-1492.
[16] ZHU H,CHEN D,XIE X,et al.Melittin inhibits lung metastasis of human osteosarcoma:Evidence of WNT/β-CATENIN signaling pathway participation[J].Toxicon,2021,198:132-142.
[17] LIN Y,ZHAN M,CHEN X,et al.Biological function of EPHB4 in the aging process of vascular endothelial cells:mtDNA molecular mechanism and MAPK/PGC-1/TFAM signaling pathway[J].Int J Biol Macromol,2024,293:138536.
[18] SHAO L,YE Q,JIA M.miR-130-3p Promotes MTX-Induced immune killing of hepatocellular carcinoma cells by targeting EPHB4[J].J Healthc Eng,2021,2021:4650794.
[19] AZEVEDO MARTINS J M,RABELO-SANTOS S H,DO AMARAL WESTIN M C,et al.Tumoral and stromal expression of MMP-2,MMP-9,MMP-14,TIMP-1,TIMP-2,and VEGF-A in cervical cancer patient survival:a competing risk analysis[J].BMC Cancer,2020,20(1):660.
[20] BAI C,MA X,WANG X,et al.Correlation between pathological features and protein expressions of TfR1,VEGF and MMP-9 in patients with osteosarcoma[J].Am J Transl Res,2022,14(7):4562-4572.
[21] DING L,LIU T,QU Y,et al.lncRNA MELTF-AS1 facilitates osteosarcoma metastasis by modulating MMP14 expression[J].Mol Ther Nucleic Acids,2021,26:787-797.